Renal function analysis method and apparatus

ABSTRACT

A method for measuring a glomerular filtration rate in a mammalian kidney comprises a source of reporter and marker fluorescent molecules. The fluorescent molecules are introduced into the blood stream of a mammalian subject. Over a period of time, a measurement of the intensities of the reporter and marker fluorescent molecules is taken. A ratio is calculated to determine the health of the subject&#39;s kidney. This method measures volume of plasma distribution based on a fluorescence of a marker molecule relative to a fluorescence of a reporter molecule.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.61/046,273, filed on Apr. 18, 2008 which is hereby incorporated byreference as if fully set forth herein.

FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

N/A

TECHNICAL FIELD

The invention relates to medical methods and devices used in conjunctionwith analyzing organ functions. More particularly, the present inventionis directed to an apparatus and method used for analyzing andquantifying function of a mammalian kidney.

BACKGROUND OF THE INVENTION

Acute kidney injury (AKI) is a serious and deadly disease processaffecting 5-10% of all hospitalized patients. The mortality rate inthese cases often exceeds 50%. AKI is independently associated withincreased mortality rates in several clinical situations, includingsubsequent to administration of radio contrast dye and cardiovascularsurgery. It is often multi-factorial in etiology, especially incritically ill patients. The relative importance of individual factorsdepends upon the underlying pathology and patient co-morbidities.

Recent data demonstrate an alarming increase in the total number ofcases of AKI. Utilizing patient claims in the Medicare 5% sample from1992-2001, Xue et al (J Am Soc Nephrol 17:1135-1142 2006) have shownthat during this time period, the incidence of AKI increasedapproximately 11.6% per year from 23.6 cases per 1,000 discharges in1992 to 63.3 cases per 1,000 patients in 2001.

In a recent study, Hsu et al (Hsu, et al., “Community-Based Incidence ofAcute Renal Failure,” Kidney Int. 2007; 72(2):208-12.) quantified theincidence of non-dialysis and dialysis AKI among members of a largeintegrated health care delivery system. Between 1996 and 2003, theincidence of non-dialysis-requiring AKI increased from 323 to 522 whilethe incidence of dialysis-requiring AKI increased from 20 to 30 per100,000 person years. Furthermore, hospital death rates were much higherin patients with AKI than in non-AKI discharges. Patients without AKIhad a 4.6% in-hospital death rate while those with primary AKI andsecondary AKI had rates of 15.2 and 32.6%, respectively. Death within 90days after hospital admission was 13.1% in discharges without AKI, 34.5%and 48.6% of patients with primary and secondary AKI, respectively. Inthis large study, the probability of developing end stage renal diseasewas 18.8% in patients with acute kidney injury as a principle diagnosisand 10.1% in patients with acute renal failure as a secondary diagnosticcode. Finally, using the data collected, it was calculated that at least22.4% of the end stage renal disease (ESRD) cases in the United Statescome from Medicare beneficiaries who had hospital acquired AKI.

These data are in agreement with observations made by Dr. Paul Eggers,director of epidemiology NIDDK, indicating a rapid increase in thepercentage and absolute number of hospitalized patients with AKI as aprimary or secondary diagnosis and in patients with chronic kidneydisease (CKD) progressing onto ESRD having had AKI as a hospitaldiagnosis.

In another study (Uchino, et al., “An Assessment of the RIFLE Criteriafor Acute Renal Failure in Hospitalized Patients,” Crit. Care Med. 2006;34(7):1913-7.) the incidence and outcomes of 20,126 hospitalizedpatients was determined in a retrospective single-center study. Of thesepatients 14.7% required ICU admission, 18% had AKI, and mortalitycorrelated with the extent of kidney injury. Finally, in a multi-centerretrospective ICU study AKI occurred in 67% of admissions and again theoverall prognosis correlated with the severity of AKI.

Clearly, the prevalence of AKI in hospitalized patients is increasing atan alarming rate. The severity of injury determines hospital outcomes,and AKI accelerates the development of chronic kidney disease andprogression of CKD to ESRD.

It is believed that glomerular filtration rate GFR is the most relevantmetric for determining the extent of AKI and progression of CKD.Reductions in the GFR secondary to kidney injury, either acute orchronic, are accompanied by increases in blood urea nitrogen (BUN) andserum creatinine levels. Currently, either serum creatinine or anequation based on the serum creatinine is used to determine a patient'sestimated GFR (eGFR). Unfortunately, these two approaches are notreliable over the full range of GFR, and neither can be used in AKI,since both muscle mass (creatinine is a breakdown product of creatine,which is an important part of muscle) and GFR determine a patient'sserum creatinine level.

Using serum creatinine as an indicator of GFR is highly patientspecific. For instance, a serum creatinine of 1.0 mg/dl is indicative ofa normal GFR (100 ml/min) in a 70 Kg (154 lb) male with normal musclemass. However, in a 50 Kg (110 lb) male with moderate muscle wasting, aserum creatinine of 1.0 mg/dl is seen even though his GFR is only 50ml/min. Formulas derived from large population studies have beendeveloped to factor in patient weight, age, sex and race. However, eventhese formulas are inaccurate and often misleading in estimating GFRbelow 20 or above 60 ml/min. Therefore, this is another reason theycannot be used in the setting of AKI.

Recent data indicate that even very small changes in kidney function, asdetermined by small total equilibrium elevations in serum creatinine,previously felt to be clinically insignificant, are now known to predictan increased mortality rate. Several recent publications have utilizedthe Risk, Injury, Failure, Loss and ESRD criteria (often called “RIFLE”criteria) to stratify patients into apparent levels of injury based onthe maximum serum creatinine obtained and the need for dialysis. Datacollected for mortality, length of hospital stay (LOS), LOS of ICU stay,hospital costs, and the need for renal replacement therapy related tothe highest stage achieved in this stratification system. These dataindicate that the severity or extent of kidney injury in AKI is animportant prognostic indicator of a patient's outcome. Furthermore,early changes in organ function predict survival in severe sepsis.

Serum creatinine determinations as a measure of GFR may also be severelylimiting because of the time it takes to reach equilibrium valuesrequired for an accurate conversion. Patients with acute renal failuredevelop an abrupt decline of their GFR; however, the magnitude of thisdecline is only apparent after several days of equilibration ifdetermined by a rising serum creatinine. For instance, if a patient wasto lose 95% of his GFR secondary to AKI, the GFR would decrease from 100to 5 ml/min rapidly, but the serum creatinine would only rise by 1mg/dl/day. This slow rise in serum creatinine limits the physician'sability to diagnose the injury for 12-24 hours after the event, and itis also not possible to determine the extent of injury for days. Thishas markedly limited the ability to conduct a therapeutic trial in AKI.Since the extent of the decline in GFR, or eventual plateau in serumcreatinine, correlates with morbidity, mortality and recovery potential,the ability to accurately determine GFR in patients with acute kidneyinjury is of great clinical importance for rapid diagnosis,stratification and timely treatment.

It is widely held that beginning therapy after 12-24 hours of AKI maylimit the success rate of any potential therapeutic agent. Therefore, asearch for a biomarker of kidney injury has intensified and is nowconsidered by many experts to be the highest priority in the field ofAKI. Potential molecules include NGAL, KIM-1, IL-18, and several others.Any one biomarker, or probably a combination of biomarkers, will serveas structural markers of injury. However, improvements sought utilizingthese structural biomarkers may not be significant because they weredeveloped using population results that may not apply to an individual.

Collection of a 24 hour urine and invasive techniques exist toaccurately determine a patient's GFR, but these are cumbersome, errorprone, expensive, time consuming, or expose the patient to radiation orradio contrast media. Also, there is no rapid and accurate measurementtechnique that can determine GFR reliably in patients with acute kidneyinjury when the serum creatinine is rising.

The liver is responsible for several activities including clearingmetabolites and toxins from the blood, making bile, lipid metabolism,drug metabolism, metabolizing many medications, storing various vitaminsand protein synthesis. Unfortunately, the liver may be diseased eitheracutely or chronically and its ability to perform various vitalfunctions may be limited. In an intensive care unit, one of the liver'smost important functions is to metabolize medications, either from theirinactive to their active state or vice versa. As a result, liver healthmay be critical to determining how much medication should be introducedinto a patient and for how long. Current methods of quantifying and/ordetecting liver function or dysfunction are generally vague andqualitative and may include jaundice, darkened urine, nausea, loss ofappetite, unusual weight loss or weight gain, vomiting, diarrhea, lightcolored stools, generalized itching, hypoglycemia, and the like.Unfortunately, these tools of detecting liver health are often identicalto signs used to detect other major health issue and are often uselesswhen diagnosing and treating a patient with multiple morbidities. As aresult, many liver diseases remain unrecognized until they reach asevere state where metabolic functions and ascites are often moredefinitive signs. As a result, a more quantitative rather thanqualitative diagnostic for liver function is needed.

The present invention is provided to solve the problems discussed aboveand other problems, and to provide advantages and aspects not providedby prior diagnostic techniques. A full discussion of the features andadvantages of the present invention is deferred to the followingdetailed description, which proceeds with reference to the accompanyingdrawings.

SUMMARY OF THE INVENTION

One aspect of the present invention is directed to a composition forintroduction into a mammalian subject's vascular system to analyze anorgan function. The composition comprises a reporter molecule and amarker molecule. The reporter molecule and the marker molecule share acommon molecular property. The reporter molecule has a reporter moleculemolecular property of a first quality, and the marker molecule has amarker molecule molecular property of a second quality which isdistinguishable from the reporter molecule molecular property firstquality. The molecular property may be chosen from the group consistingof molecular weight, molecular size, molecular shape, molecular charge,compound, and radio frequency.

When the molecular property is molecular weight, the reporter moleculemay have a first molecular weight and the marker molecule may have asecond molecular weight. The first molecular weight may be less than thesecond molecular weight, and may be substantially less. The firstmolecular weight may be of a magnitude wherein the reporter molecule isfiltered by a properly functioning mammalian kidney. The secondmolecular weight may be great enough to resist filtration of the markermolecule by a mammalian kidney. Optionally, the first molecular weightmay be of a magnitude wherein the reporter molecule is readily filteredby a properly functioning mammalian kidney, while at the same time, thesecond molecular weight is great enough to resist filtration of themarker molecule by a mammalian kidney. The first molecular weight may bechosen from a group of ranges consisting of 1 kD to 500 kD, 3 kD to 150kD, 10 kD to 150 kD, 10 kD to 70 kD, and 20 kD to 70 kD. Alternatively,the first molecular weight may be less than 500 kD, between 1 kD and 500kD, 3 kD and 150 kD, between 3 kD and 70 kD, between 3 kD and 20 kD, orabout 5 kD.

The reporter molecule may have a first fluorescent characteristic, andthe marker molecule may have a second fluorescent characteristic. Thesefluorescent characteristics may not be equal, e.g. having differingwavelengths. The first fluorescent characteristic may be a firstfluorescence excitation wavelength and a first fluorescence emissionwavelength. The second fluorescent characteristic may be a secondfluorescence excitation wavelength and a second fluorescence emissionwavelength. The first and second fluorescence excitation wavelengths andthe first and second fluorescence emission wavelengths may be different,unequal, or distinguishable.

The reporter and marker molecules may be dextrans. The reporter moleculemay be a sulphorhodamine 101 dextran. The marker molecule may be alarger undefined dextran that is not filtered by a mammalian kidney. Thereporter molecule and the marker molecule may be dextrans conjugatedwith fluorescein. Additionally, the reporter molecule fluorescein mayhave a fluorescence excitation wavelength that is not equal to afluorescence excitation wavelength of the marker molecule.

Alternatively, the reporter molecule and the marker molecule may bedextrans conjugated with different fluorophores. Additionally, thefluorescent reporter molecule may have a fluorescence excitationwavelength that is not equal to a fluorescence excitation wavelength ofthe marker molecule.

The reporter molecule may be a fluorescein isothiocyanate-inulin.

The marker molecule may have a glomerular sieving coefficient of about0.

The marker molecule may not be not secreted, reabsorbed, or filtered bya mammalian kidney.

The marker molecule may not be capable of passing through a glomerularfiltration barrier, and the reporter molecule may be capable of passingthrough a glomerular filtration barrier.

A second aspect of the present invention is directed to an apparatus foranalyzing an operating condition of a mammalian kidney. The apparatuscomprises a source of fluorescent molecules, a means for introducing thefluorescent molecules into a vascular system, a means for measuring thefluorescent molecules within the vascular system, and a means forreporting the measured fluorescent molecules within the vascular system.The means for introducing may include a catheter. The means formeasuring may include an optic fiber in communication with a detector.The means for reporting may include determining an intensity ratiobetween two or more fluorescent molecules measured within the vascularsystem. The source of fluorescent molecules may comprise a plurality offluorescently conjugated molecules.

A third aspect of the present invention is directed to an apparatus foranalyzing an operating condition of a mammalian kidney. The apparatuscomprises an optical means providing a first excitation wavelength to afirst fluorescent molecule and a means for measuring an emission fromthe first fluorescent molecule in response to the first excitationwavelength.

The optical means may emit a second excitation wavelength to a secondfluorescent molecule, and the apparatus may further comprise a means formeasuring an emission from the second fluorescent molecule in responseto the second excitation wavelength.

The apparatus of the third aspect of the invention may further comprisea means for calculating a ratio of the emission from the firstfluorescent molecule to the emission from the second fluorescentmolecule. The apparatus may still further comprise a means for reportingthe ratio.

A fourth aspect of the present invention is directed to an opticalapparatus for measuring a relative amount of a plurality offluorescently conjugated glomerular filtration rate molecules within avascular system. The apparatus comprises a source of a first fluorescentexcitation wavelength, a delivery optical path along which thefluorescent excitation wavelength passes, an excitation site to whichthe fluorescent excitation wavelength is delivered, a return opticalpath along which an emitted fluorescence signal passes, and a means fordetecting an intensity of the emitted fluorescence signal. Thisapparatus may further comprise a source of a second fluorescenceexcitation wavelength.

The means for detecting may be chosen from a group consisting of a photomultiplier tube, a photo detector, a solid state detector, and acharge-coupled device.

The excitation site may include a fiber optic cable.

A fifth aspect of the invention is directed to an optical apparatus formeasuring a relative amount of a plurality of fluorescent glomerularfiltration rate molecules within a vascular system. The opticalapparatus comprises a source of a first fluorescent excitationwavelength, a source of a second fluorescent excitation wavelength, adelivery optical path along which the first and second fluorescentexcitation wavelengths pass, an excitation site to which the first andsecond fluorescent excitation wavelengths are delivered, a returnoptical path along which a first emitted fluorescence signal and asecond emitted fluorescence signal pass from the excitation site, afirst means for detecting an intensity of the first emitted fluorescencesignal; and a second means for detecting an intensity of the secondemitted fluorescence signal.

The optical apparatus of the fifth aspect of the invention may furthercomprise a first lens for focusing at least one of the first or secondfluorescent excitation wavelengths onto the excitation site. A secondlens may focus at least one of the first or second emitted fluorescencesignals onto one of the first or second means for detecting. A thirdlens may focus the other of the first or second emitted fluorescencesignals onto the other of the first or second means for detecting. Afirst condenser lens may be provided for minimizing an aberrationassociated with the first fluorescent excitation wavelength. A secondcondenser lens may be provided for minimizing an aberration associatedwith the second fluorescent excitation wavelength. A first dichroicfilter may be positioned within the delivery optical path. A seconddichroic filter may be positioned within the return optical path. Athird dichroic filter may be positioned within the optical devicebetween the delivery optical path and the return optical path.

A sixth aspect of the present invention is directed to a catheter foruse in analyzing an operating condition of a kidney. The cathetercomprises a tubular main member having a proximal end opposite a distalend and defining a passageway and a fiber optic cable extensible fromthe distal end. The catheter may further comprise an introducerconnected to the tubular member at one end and having an opposite endinsertable into a vascular system, and/or an insertion tool. A length ofthe fiber optic cable may be fluid sealed within the insertion tool. Theinsertion tool may include a first tubular member slidable within asecond tubular member. The fiber optic cable may be held captive by aportion of the first tubular member. The insertion tool may be joined toa port of the tubular main member wherein the fiber optic cable passesthrough the insertion tool and into the tubular main member. The tubularmain member may be joined to the introducer by a connector. The fiberoptic cable may be extensible from the introducer upon relative movementbetween the first and second tubular members.

The first and second tubular members may be fluidly sealed.

The fiber optic cable may include a bend on a distal end insertable intoa vascular system.

A seventh aspect of the present invention is directed to a catheter foruse in analyzing an operating condition of a kidney. The cathetercomprises a fiber optic cable, a fiber optic insertion tool about alength of the fiber optic cable having a first tubular member sealed toa second tubular member and capable of relative movement therewith, thefiber optic cable held attached to a portion of the first tubular membersuch that movement by the first tubular member transfers movement to thefiber optic cable, and a tubular main body sealed to the insertion tool,the fiber optic cable passing through a passageway in the tubular mainbody and extensible therefrom upon relative movement between the firstand second tubular members.

The catheter may further comprise an introducer connected to the tubularmain body. The introducer is insertable within a vascular system, andthe fiber optic cable extensible therefrom.

The fiber optic cable may have a bend at one end. The one end isinsertable within a vascular system.

An eighth aspect of the present invention is directed to a method ofmeasuring a glomerular filtration rate in a mammalian kidney. The methodcomprises the steps of: providing a plurality of first fluorescentmolecules; providing a plurality of second fluorescent molecules;introducing the first fluorescent molecules and the second fluorescentmolecules into a blood stream of a mammalian subject; exciting the firstfluorescent molecules with a first fluorescence excitation wavelength togenerate a first fluorescence emission signal having a firstfluorescence emission wavelength and exciting the second fluorescentmolecules with a second fluorescence excitation wavelength to generate asecond fluorescence emission signal having a second fluorescenceemission wavelength; measuring an intensity of the first fluorescenceemission signal and an intensity of the second fluorescence emissionsignal subsequent to the introducing step; and calculating a ratio ofthe first fluorescence emission signal to the intensity of the secondfluorescence emission signal. The measuring and calculating steps may beperformed at predetermined intervals and reported in at leastsubstantially real time.

A ninth aspect of the present invention is directed to a method ofmeasuring a glomerular filtration rate in a mammalian kidney. Thismethod comprises the steps of: providing a fluid comprising a pluralityof reporter molecules and a plurality of marker molecules in apredetermined ratio; introducing the fluid into a vascular system of asubject; and measuring a characteristic of each of the reporter andmarker molecules after an elapsed time duration within the vascularsystem of the subject. This method may further comprise the steps of:calculating a ratio of the reporter molecule characteristic and themarker molecule characteristic subsequent to the measuring step; andreporting the ratio. The reporting step may be performed at a pluralityof elapsed time durations. The calculating and reporting steps may beperformed in at least substantially real time.

The reporter molecules may be capable of passing through a glomerularfiltration barrier. The marker molecules may be less capable than thereporter molecules of passing through a glomerular barrier. The reportermolecules and the marker molecules may be fluorescent molecules. Thereporter molecules and the marker molecules may comprise dextrans. Themarker molecules may have a greater molecular weight than the reportermolecule. The reporter molecule may have a molecular weight between 1 kDand 150 kD. The marker molecule may have a molecular weight greater than100 kD.

A tenth aspect of the present invention is directed to a method ofmeasuring a glomerular filtration rate in a mammalian kidney. Thismethod comprises the steps of: providing a source of light having aknown wavelength; exposing a fluorescent molecule to the light sourcewherein the fluorescent molecule is excited within a vascular system ofa mammalian subject for a predetermined time duration; and measuring acharacteristic of the excited fluorescent molecule, the characteristichaving a correlation to a condition of the vascular system.

Other features and advantages of the invention will be apparent from thefollowing specification taken in conjunction with the followingdrawings.

BRIEF DESCRIPTION OF THE DRAWINGS

To understand the present invention, it will now be described by way ofexample, with reference to the accompanying drawings in which:

FIG. 1 is an illustration of an apparatus of the present inventionutilizing a method of the present invention;

FIG. 2 is a series of micrographs showing renal clearance of a smallmolecular weight dextran in a normal rat as visualized by intravital2-photon microscopy. Micrographs taken from a time series reveallocalization of both a small FITC-inulin (5.5 kD) (lower series ofmicrographs) and a large 500 kD Texas Red® dextran (upper series ofmicrographs) within the capillaries (CAP). The inulin is rapidlyfiltered into the proximal tubular lumen (PT lumen) resulting in asteady decrease in fluorescence signal over time (panels E, F, G, & H).In contrast, the 500 kD dextran is not cleared and its signal remainsconstant within the capillaries (panels A, B, C, & D). The fluorescenceseen in the PT lumen in Panel A, B, & C is not clearance of the 500 kDdextran but bleed through emissions from the FITC-inulin. The currentapproach of sequential excitation and acquisition with separate LED'swill minimize this bleed through phenomenon;

FIG. 3 is a series of micrographs showing the intensity ratio ofFITC-inulin to the 500 kD Texas Red® dextran with the 500 kD Texas Red®dextran staying in the blood stream a longer time after dye infusion dueto the larger molecular size;

FIG. 4 is a plot of the intensity time-series of the 500 kD FITC dextranmeasured from a blood vessel following a bolus infusion up to 60minutes;

FIG. 5 is a plot of the intensity ratio of a bolus injection of 20 kDFITC dextran to 500 kD Texas Red® dextran as a function of time alongwith the result of a least square fit;

FIG. 6 is a comparison of plots between using a ratio technique anddirectly using intensity for measuring plasma clearance;

FIG. 7 is a plot showing the kidney vascular plasma intensity ratiosresulting over time from two rats after bolus infusion, one with amixture of 3 kD FITC dextran and 500 kD Texas-Red dextran and the otherwith 20 kD FITC dextran and 500 kD Texas Red® dextran;

FIG. 8 is a plot showing liver vascular plasma intensity ratios overtime from two anephric rats, one injected with a mixture of 10 kD FITCdextran and 500 kD Texas Red® dextran, the other with a mixture of 20 kDFITC dextran and 500 kD Texas Red® dextran;

FIG. 9 is a block diagram a diagnostic apparatus of the presentinvention;

FIG. 10 is a model of the apparatus of FIG. 9;

FIG. 11 is a plot of excitation and emission spectra of FITC, rhodamineand Texas Red® dyes;

FIG. 12 is a block diagram of a single channel optical system;

FIG. 13 is a block diagram of a two channel optical system;

FIG. 14 is an exploded view of the two channel optical system of FIG.13;

FIG. 15 is a block diagram of a two compartment model;

FIG. 16 is an illustration of a dissembled catheter having an opticalfiber; and

FIG. 17 is an illustration of the catheter of FIG. 16 assembled andinserted within a patient's vein.

DETAILED DESCRIPTION

While this invention is susceptible of embodiments in many differentforms, there are shown in the drawings and will herein be described indetail preferred embodiments of the invention with the understandingthat the present disclosures are to be considered as exemplifications ofthe principles of the invention and are not intended to limit the broadaspect of the invention to the embodiments illustrated.

The inventors have found that a combination of both structural andfunctional markers of AKI presents a high level of clinical utility indiagnosing kidney function and kidney-related diseases. Thus, oneobjective of the present invention is to provide tests for analyzing andquantifying organ function and physiological parameters that have beendifficult or impossible to measure in the past. The present inventionfocuses on a method and device for rapid detection of acute kidneyinjury and chronic kidney-related diseases. This development utilizestechnology developed by and licensed from the Indiana Center forBiological Microscopy. Such technology is described in U.S. ProvisionalPatent Application No. 60/672,708, PCT Application No. US2006/014576,published as WO/2006/113724, and U.S. application Ser. No. 11/911,895,which are hereby incorporated by reference as if fully set forth herein.Specifically, figures of the apparatuses shown in FIGS. 6-9 ofWO/2006/113724 and the descriptions of same at the paragraphs numbered96 to 104 are directed to the technology utilized.

In early animal studies, this technology has proven efficacious inproviding accurate and rapid measurement of the true GlomerularFiltration Rate (GFR)—the rate by which the kidney is able to filterwaste products from the blood stream. While the need for diseasediagnostics varies according to the specific disease, in kidney disease,GFR is the primary clinical indicator of injury, disease progression, orrecovery.

GFR measures the amount of plasma filtered through glomeruli within agiven period of time. It is clinically the most widely used indicator ofkidney function. Physicians routinely use it for both diagnostic andtherapeutic decisions. In fact, the National Kidney Foundation has nowdivided chronic kidney disease patients into five groups (I-V) basedupon their estimated GFR (eGFR). This has assisted clinicians inrecognizing and understanding the severity of the kidney disease inpatients. It has also allowed for the initiation of appropriatetherapies based on the patient's baseline GFR.

A variety of techniques such as radioactive and non-radioactive contrastagents, as well as radiographic renal imaging, can measure GFR rapidly.Plasma clearance techniques are based on measuring the plasma clearanceof GFR marker molecules. By using radioactive markers, such as[51]Cr-EDTA or [99]m Tc-DTPA ([99]m Technetium diethylene triaminepentaacetic acid), it has been reported that plasma clearance and GFRcould both be determined independently using a radiation detector. Usingradioactive GFR markers, such as [51]Cr-EDTA and [99]mTc-DTPA[99]m-Technetium diethylene triamine pentaacetate), in conjunction witha radiation detector, one can monitor GFR in patients with acute kidneyinjury at rates close to real-time. The measured plasma clearance showsexcellent correlations with GFRs simultaneously measured using thestandard method with urine collection. However, the use of radioactiveGFR markers and the clinical difficulties in administering this testmake this method unattractive. By using a fluorescent GFR marker, suchas FITC-inulin, with a bolus intravenous infusion followed with drawingblood samples at multiple time points, one can accurately determine GFR.Potentially, with the development of a suitable contrast agent, magneticresonance imaging (MRI) techniques can be very useful for providingkidney functional diagnostics. The downside of using such technologiesis the low accessibility, associated high cost, difficulty repeating thestudy and the need to move the patient for the study.

Similarly, the plasma concentration of non-radioactive markers, e.g.iothalamate, determined by standard methods, such as high-performanceliquid chromatography (HPLC), has also been used to evaluate renalfunction in critically ill patients. Such plasma clearance based GFRmeasurement techniques have been reported to have good time resolutionin detecting changes of renal function in patients with severelyimpaired renal function. By using bolus infusion of a single fluorescentGFR marker, FITC-inulin, GFR has been determined by sequentiallymeasuring the fluorescence signals in the blood samples drawn as afunction of time after infusion. The inventors have expanded upon andenhanced this approach offering improved accuracy, rate ofdetermination, and reduced exposure to potentially toxic radioactivemolecules.

Inulin, a small fructose polymer that is filtered, and cleared from thebody only by glomerular filtration, is a reference standard GFR marker.Other non-radioactive markers (such as iothalamate, iohexol,polyfructosan) and radioactive ones (such as [125]I-iothalamate and[51]Cr-EDTA) are also commonly used.

In clinical practice, endogenous markers such as serum creatinine andcystatin C are routinely used to estimate GFR, since the production andtubular reabsorption rates of these molecules vary significantly fromdifferent individuals. Cystatin C has received recent attention as asuperior endogenous serum marker of GFR, compared to serum creatinine,as it is elevated up to a day earlier than creatinine in an ICUpopulation with AKI.

The inventors have developed a minimally invasive device for directmeasurement of GFR in mammalian subjects, such as humans, using amulti-photon microscopy method, preferably a two photon microscopymethod. The method relies on reading two fluorescent molecules attachedto different size dextran molecules. Dextran is a complex, branchedpolysaccharide made of many glucose molecules joined into chains ofvarying lengths (from 3 to 2,000 kD). Thus, another objective of thepresent invention is to provide both a method and apparatus using acatheter based fiber optic probe to read the fluorescent markers. Thiscatheter can be placed into a vascular system, e.g., an arm vein of amammalian patient, to allow the concentration of fluorescent markers tobe monitored in real time, providing a direct measurement of GFR.

A rapid and accurate measurement of GFR in an early stage of acutekidney injury is important for diagnosis, stratification of extent ofinjury and therapeutic purposes. An advantage of the present inventionis that it will rapidly identify and determine the extent of injuryallowing for early treatment, including dialysis initiation, as well asenrollment and stratification for clinical studies. It could also beused to determine the effect of a clinical maneuver on GFR, such asvolume resuscitation. Therefore, this technical advance is of majorclinical importance, especially in high risk patients where intensesurveillance is necessary for early diagnosis, injury stratification anddetermination of therapeutic potential.

The inadequacies of methods currently clinically used for estimating GFRare established both in literature and in practice. While progress isbeing made to identify biomarkers for detecting presence of injury,little progress has been made in finding a functional marker that ispractical enough for broad acceptance. The inventors' method representsa true advancement in the ability to accurately quantify and track thedegree of kidney function with near real-time efficiency. The inventorshave also developed a device that is easy to operate in a busy medicalenvironment—a critical adoption barrier in medical technology.

The optical technique developed by the inventors is based on plasmaclearance measurements of a fluorescent bioreporter molecule and allowsfor the rapid, frequent, and safe evaluation of GFR. To further validatethe values, other standard GFR tests, including but not limited toinulin clearance, may be performed. Upon comparison of these values, acorrection factor may be applied to the data obtained using this novelmethod if needed.

Referring to FIG. 1, an apparatus 10 which incorporates a method of thepresent invention is illustrated. The apparatus 10 comprises a source ofa GFR measurement composition 100, a kidney fluorescent detector 200,and a catheter 300. The GFR measurement composition 100, which comprisesa plurality of reporter molecules and a plurality of marker molecules,is introduced into the blood stream of a human subject 20 via thecatheter 300. The fluorescent detector 200 monitors the level of the GFRmeasurement composition within the blood stream and reports an operatingcondition of the human subject's kidney in at least substantially realtime. This apparatus measures volume of plasma distribution based on afluorescence of a marker molecule relative to the fluorescence of areporter molecule. “Substantially real time” is intended to encompassthe duration elapsed between measurement of the levels of the reporterand the marker within the blood stream, calculation of the operatingcondition of the kidney, and reporting of that condition. It iscontemplated by the inventors that this elapsed time will be very nearreal time as to be negligible in relation to the prior techniquesdiscussed above.

The GFR Measurement Composition

By utilizing intravital multi-photon microscopic imaging of the kidney,the inventors have quantified glomerular filtration and tubularreabsorption processes independent of each other. The inventors havedeveloped ratiometric imaging techniques permitting quantitativeanalysis of fluorescence signals within local regions of the kidneyusing multi-fluorescent probe experiments. To measure GFR by plasmaclearance, the inventors use a fluorescent GFR reporter molecule, e.g.FITC dextran, together with a large different fluorescent markermolecule that does not pass through the glomerular filtration barrier.This large fluorescent marker serves to quantify the plasma volume ofdistribution in the vascular space and allow for the ratiometrictechnique.

The inventors have been able to quantify plasma clearance of thefluorescent GFR marker by examining the ratio of fluorescenceintensities of the two molecules from within the blood vessel regions ofthe image. GFR can be rapidly determined using this ratio technique.This method has been tested in a number of animal models. Since thefluorescent signals are being measured from within the blood vessels toquantify the kinetics of plasma clearance, the ratio signal of the twofluorescent molecules is independent of the body location where themeasurement is performed.

To measure GFR accurately, the inventors have determined that the idealGFR marker molecule should be stable within the vascular compartmentduring the study and have a glomerular sieving coefficient (GSC) of 0.0,be retained within the vasculature, and it should not be, or shouldsubstantially not be, secreted, reabsorbed, or filtered within thekidney and may have a molecular weight greater than 100 kD.“Substantially” as used here is limited to ±5%. Satisfying theseconditions, the GFR would be equal to the urinary clearance of thereporter after its intravenous infusion. In theory, one could use a GFRreporter with any known GSC that is greater than 0. The preferablemarker molecule is a sulphorhodamine 101 having a molecular weightgreater than 100 kD.

FIG. 2 contains several fluorescence intensity images of the kidney froma live and healthy male rat. These images were taken as function of timeafter a bolus intravenous infusion of a dye mixture containing aFITC-inulin (5.5 kD) and 500 kD dextran labeled with a sulforhodamine101, i.e. Texas Red®. The fluorescence intensity signal from FITC-inulinis shown in the lower series of micrographs, and the 500 kD Texas Red®dextran intensity is shown in the upper series of micrographs. At about12 seconds after dye infusion, the fluorescence intensity was seen inboth the capillaries of the kidney and in the proximal tubule (PT)lumen. The variations in the blood vessel over time indicate that boththe FITC-inulin and 500 kD Texas Red® dextran were in these bloodvessels. At 50 seconds, the FITC-inulin was already decreasing inintensity in the capillary and in the PT lumen as a result of immediateplasma clearance (glomerular filtration) of this molecule. This was nottrue for the red 500 kD dextran where the capillary intensity wassimilar to the 12 second value. A red blood cell (RBC) appears as a darkobject as it excludes dye. At 100 and 200 seconds the FITC-inulinintensity continued to decrease in the capillary and in the PT lumen asfiltration continued to remove it from the body. This again was not truefor the 500 kD Texas Red® dextran which did not change in intensityduring this time interval as it was not filtered. Consequently, therelative strength of the intensity from the blood vessels increasesindicating a relative increase in the 500 kD Texas Red® dextran toFITC-inulin concentration ratio due to plasma clearance of theFITC-inulin. This type of time-series image collection contains dynamicinformation about a given molecule passing through the glomerularfiltration barrier of the kidney, and becoming part of the filtrate.This provides the basis for the inventors' measurement of plasmaclearance rates and GFR.

To quantify molecular filtration dynamics, the inventors used theintensity ratio of the FITC-inulin and the 500 kD Texas Red® dextran(See FIG. 3). The intensity ratio from the blood vessels in FIG. 3changes over time with a change of relative concentrations of the twodyes. Since the 500 kD dextran molecules are minimally cleared from thevascular culture, not by the kidneys, due to its large size, it remainsstable in the plasma for a long time after infusion. Typically, therewas no noticeable intensity drop from the 500 kD dextran within the timeperiod following a dye infusion (anywhere between 5-30 minutes). Thisresulted in a decreasing intensity ratio visualized over time. It isthis type of ratio that greatly minimizes the problems with usingfluorescence intensity as a read out for biological studies.

The inventors have also used a 500 kD fluorescent dextran for similarstudies in order to further minimize filtration and extend the dye'splasma survival time. FIG. 4 is an example of the intensity time-seriesof the 500 kD FITC dextran measured from a blood vessel following abolus infusion up to 60 minutes. The initial intensity spike (see inlet)was due to dye injection and fast distribution of the dye molecules intothe whole plasma volume. It did not show significant intensity drop forthe rest of the curve. Effectively, the decrease of the fluorescenceintensity ratio of labeled inulin to labeled 500 kD dextran correlateswith the concentration decrease of the labeled inulin.

FIG. 5 is a plot of the intensity ratio of a bolus injection of 20 kDFITC dextran to 500 kD Texas Red® dextran as a function of time alongwith the result of a least square fit. Each data point in FIG. 5 was theaverage ratio value of the same region from a blood vessel extractedfrom an image time-series (such as the images shown in FIG. 3). The datapoints were plotted every 0.5 seconds up to 200 seconds. The decayoccurred in two phases, the initial phase and the clearance phase (orthe filtration phase/elimination phase). The gradual increase of theinitial phase was due to relative dye distributions and accumulations inthe kidney following IV injection. The highest point (around 12 seconds)of the curve marks the starting point of the clearance phase andcorrelates with the beginning of the appearance of FITC-inulin in theproximal tubule.

The data points of the clearance phase fit well with a singleexponential. The inventors obtained a 20 kD FITC dextran plasmaclearance rate constant, k, of 0.00458 (s⁻¹) (using 95% confidencelimits).

Following a bolus infusion of GFR reporter molecules, the plasmaconcentration of the GFR reporter molecules decreases as a function oftime due to renal clearance. By acquiring plasma samples at differenttime points, one can either directly calculate or perform least squarefit of the time trace to retrieve the plasma clearance rate constant(k). GFR can then be determined according to the equation:

GFR=kV _(d)  (1)

where k is the plasma clearance rate and V_(D) is the volume ofdistribution into which the GFR marker is diluted. GFR measured usingthis technique has been validated in patients with stable renal functionas well as in rodents and proven to be accurate and correlated well withwhat was measured using other methods.

A comparison between using the intensity ratio and directly using theintensity value of a 3 kD FITC conjugated dextran (3 kD FITC dextran)for measuring the clearance rate is shown in FIG. 6. The chiefdifferences are significantly less noise and better identification ofdistribution phase.

The intensity fluctuations of the 3 kD FITC dextran alone were quitesignificant (FIG. 6-B). Consequently, the fitting result of theclearance rate constant k contained larger errors and was less defined.In contrast, the intensity ratio (between 3 kD FITC dextran and a 500 kDTexas Red® dextran) had significantly less noise, and the measuredclearance rate constant k was much better defined with only 3% error.This was partially because fluorescence intensity is typically verysensitive to even a slight change in microscope focus and movement ofthe sample. The intensity ratio, on the other hand, is insensitive tominor changes in imaging depth and motion. The inventors are focusing onthe fluorescence signals from the blood. Furthermore, the intensitysignal of a dye from the blood can change when the blood flow ratechanges. However, the relative intensity ratio between two moleculesdoes not change even when the blood flow rate or blood volume changes(assuming there is no clearance). This is because both dye molecules arepresent in the blood and move together. The method developed by theinventors limits this problem.

The separation between the initial dye distribution and the clearancephase is well-defined using the intensity ratio. When using theintensity of a single dye alone, it is more difficult to determine atwhat time point the clearance phase begins. The highest data point inthe intensity curve typically does not correlate in time with theappearance of the smaller molecule in the proximal tubule lumen.Therefore, the dye distribution and the filtration phases are convolutedin the intensity only curve. Using multi-photon microscopy approachesallow such correlations and is highly beneficial.

It is believed that purity, in terms of size distribution or molecularweight, of the dextrans is vitally important. In addition, thedistribution of molecular weight plays an important role in how well GFRcan be measured. Even though dextrans are widely used in medicalapplications, these previous applications did not require the morestringent size control needed for use in the present invention.

Referring to FIG. 7, a plot of intensity values obtained from two ratsafter bolus infusion is illustrated. One rat was infused with a mixtureof 3 kD FITC dextran and 500 kD Texas Red® dextran. The second rat wasinfused with 20 kD FITC dextran and 500 kD Texas Red® dextran. The plotshows a rapid decay with the 3 kD/500 kD fluorescence ratio curve. Thisindicates a fast clearance with movement into the interstitial space.However, a substantial part of it is due to non-renal plasma clearanceas seen from 10 kD dextran data of liver imaging illustrated in FIG. 8.

FIG. 8 was generated from a pair of anephric rats (with both kidneysremoved). One of the rats was injected with a mixture of 10 kD FITCdextran and 500 kD Texas Red® dextran. The other rat was injected with amixture of 20 kD FITC dextran and 500 kD Texas Red® dextran. The 10kD/500 kD ratio curve shows that there is clear evidence that non-renalplasma clearance of 10 kD dextran is still substantial. Meanwhile, the20 kD dextran, which can be filtered by glomeruli, shows minimalnon-renal plasma clearance. Therefore, it can be used to determine GFR.

Additionally, smaller molecules of 3 kD to 5 kD as reporter molecules inconjunction with a two compartment kinetic model can be used to measureorgan function. Thus, the inventors have determined that the preferredmolecular weight of the filtered molecule to be within the range of 3 kDto 500 kD, more preferably 3 kD to 150 kD, still more preferably 3 kD to150 kD, still more preferably 3 kD to 70 kD, still more preferably 3 kDto 5 kD, and most preferably on the order of 5 kD, or any range orcombination of ranges therein. The method also contemplates the use ofknown common sizes such as 10 kD and 500 kD dextrans as well less commonsizes 20 kD, 70 kD and 150 kD. An amino fluorescein dextran ispreferred.

Fluorescent Detector

The fluorescent detector 200 includes software for reading and reportingdata, a user interface 202 to control the apparatus 10 and reviewresults, and an apparatus for sending and receiving fluorescent signals204 (see FIGS. 9, 10, and 12-14). This unit 200 is designed to becompatible with a standard IV pump stand, or it can be operated on atable top. It incorporates a battery backup system that is capable ofrunning for 2 hours without connection to AC power.

The user interface 202 is capable of being used by any clinician. Itincludes touch screen technology for most of the software userinterface. This provides flexibility in how the data is shown to theclinicians.

Based on the body of work done to perfect the ratio technique usingmulti-photon microscopy, the inventors determined that a fiber opticcatheter placed in the blood stream of a subject would be capable ofmeasuring the fluorescent molecules. The current method of usingmulti-photon microscopy is responsible for generating much of thevariation due to the drop off in fluorescence intensity as the tissue ispenetrated more deeply. Using a fiber optic catheter as disclosed hereinwill eliminate these variations since the measurements will be taken inreal time, or substantially real time, directly in the blood. The fiberoptic catheter is explained in more detail below.

FIGS. 9 and 10 illustrate a two channel apparatus 204 using a singlemulti-colored LED 206 (light emitting diode) as a light source. Anobjective of this apparatus 204 is to determine how much fluorescentsignal would be returned from a fiber optic element 210. FIGS. 9 and 10show both a diagram and computer model of the optical system used inthis device. The apparatus includes photo multiplier tubes (PMT) 208 a,bas detectors, since these devices have well-known characteristics.Alternatively, the detector may be a photo detector, a solid statedetector, a charge-coupled device, or any other equivalent devicewithout departing from the spirit of the invention. This apparatus mayhave one or more power supplies 207,209, and/or controllers, forproviding power to the LED 206 and PMTs 208 a,b.

An optical path 212 focuses the light from an LED source 206 through aselection of band pass 216 and dichroic filters 220, then onto the fiberoptic element 210. An excitation light is then passed down the fiberoptic element 210 into a test solution chosen to simulate theapproximate level of fluorescent dextrans in a blood stream. The fiberoptic element 210 is generally a fiber optic cable in the range of 0.5to 1 mm in diameter or even smaller.

Once excited, a small portion of the fluorescence signal then passesback through the fiber optic element 210. The signal then passes througha focusing lens 224, dichroic beam splitter 228 and band pass filter 232before landing on the cathode of the PMT 208 a.

An easily detectable fluorescent signal is measured from the PMT 208 a,bfor fluorescein dye. This dye has an excitation peak of about 494 nm andemits light in a broad band of wavelengths centered on 519 nm.Fluorescein dye is only one example of a marker dye. A rhodamine dye mayalso be used; however, the LED source 206 must have sufficient intensityto excite the rhodamine dye. The spectral response of fluorescein,rhodamine and Texas Red®, can be seen in FIG. 11.

The emergence of white LEDs based on adding a phosphor to the LED diemay be used in the present device 200, but the narrow spectral bandwidthassociated with standard LEDs is superior for reducing background light.The intensity of the light source and how efficiently energy can bedelivered to the fiber optic 210 is critical.

Laser diodes may be used as a substitute for LEDs. The laser diodeprovides additional light energy which may allow a reduction in theconcentration of dye markers in the blood stream. However, most of thewavelengths available are not ideal for the preferred fluorescentmolecules of fluorescein and sulforhodamine 101.

LEDs from several vendors have been evaluated. Several LEDs meet theneeds of the apparatus. These LEDs provide the best flux density perunit area and work well with the filters providing excellent eliminationof off wavelength background.

For fluorescein, a LED490-03U made by ROITHNER LASERTECHNIK GmbH ofAustria may be chosen. This LED has a peak wavelength of 490 nm forfluorescein excitation. This LED is rated at 1.2 mw. Alternatively, forfluorescein, an XREBLU-L1-0000-00K01 LED made by Cree Inc. of Durham,N.C. is preferable. This part is a high power surface mount LED withgood thermal characteristics. The peak wavelength for this applicationis 485 nm with a minimum flux output of 30.6 lumens. A surface mountpart that can be sorted to have similar characteristics may besubstituted for this part.

For sulforhodamine 101 excitation, an 828-OVTLOILGAAS from OPTEK, havingdistribution in North America and throughout the world, with a peakwavelength of 595 nm, may be used. This surface mount LED has a higherflux density, so it can be run at lower power settings to minimizewavelength thermal drift. The target output power for the 1 mm fiberoptic will be about 50 microwatts. For sulphorhodamine 101 excitation,an XRCAMB-L1-0000-00K01 LED from Cree Inc. of Durham, N.C. ispreferable. LEDs of this type can be sorted for peak wavelength over therange of 585 nm to 595 nm. A peak output of 590 nm has been chosen forthe application. These are high power surface mount LEDs with goodthermal characteristics. The luminus flux output of this LED is also30.6 lumens.

Filter selection is critical to performance of this system. Since thefluorescence signal returning through the fiber optic 210 will be manyorders of magnitude below the excitation energy, filter blocking andbandpass characteristics are critical to proper performance. Thefluorescent markers which have been used in microscopy and otherapplications for many years are well known in the art. Thus, excellentfilter sets are available from a variety of manufacturers such asSEMROCK of the United States. These filters are ideal for thisapplication.

Two additional apparatuses for sending and receiving fluorescent signals204 have been contemplated by the inventors. These apparatuses areillustrated in FIGS. 12 and 13 and are aimed at improved opticalgeometry. A single channel apparatus is illustrated in FIG. 12. Oneobjective of the single channel device is to improve the signal tobackground ratio and determine the target signal strengths for thefluorescently tagged dextrans in whole blood. An improved opticalgeometry has significantly reduced the background levels over an orderof magnitude. This new optical geometry and fiber coupling has providedus with a 30 to 1 signal to background ratio.

FIG. 12 is a block diagram of a single channel optical system. Thesingle channel device shown in FIG. 12 uses a simple optical design.Light from a 490 nm LED 206 is relayed through a band pass dichroicfilter 220, then focused onto the fiber optic surface mount adaptor(SMA) connector 244. A simple condenser lens element 240 is used tominimize spherical aberrations that would limit ability to focus ontothe small 0.5 to 1 mm fiber optic target 210. The fiber lens 224 worksas both a final focusing element for the source light and the initialcollimator for the fluorescent emission. The fluorescent emission lightis relayed back through the dichroic filter 220 and refocused onto thePMT 208. Simple bi-convex lenses 248 are used for this since the targetsize on the PMT 208 is not critical, and a FITC emission filter isprovided as a band pass filter. Close attention is given to stray lightand reflections in this system by utilizing good light absorbing coatingmaterials in the component construction.

Referring to FIGS. 13 and 14, a two channel optical system isillustrated. Similar components to those chosen in the single channeldesign are used in the two channel design. The main differences are anadditional dichroic filter within holders 254 a,b and spaced from a mainblock 255 by spacers 256 a,b used to combine light from the 490 nm LED206 a and 595 nm LED 206 b together. Each source utilizes its owncondenser lens assembly 240 a,b and band-pass filter 216 a,b, a 595 nmfilter and a 490 nm filter respectively, within holders 242 a,b. Thelight beams from the LEDs 206 a,b are then relayed through a specialdual band dichroic filter 252 before being focused by the lens 224 ontothe fiber coupler 244, specifically the fiber optic target 210. Thisdual band filter is readily available from SEMROCK. The emission fromboth fluorescent molecules then travels back through the fiber opticcable 210. The fiber lens 224 is attached to main block 255 withinholder 258 and ring 262 and is used to collimate this light for relayback through the dual band dichroic filter 252 and then split to theappropriate PMT 208 a,b using a final emission dichroic filter 228. EachPMT assembly 208 a,b has a final focusing lens 248 a,b and an emissionfilter 232 a,b, preferably a FITC emission filter and a sulphorhodamine101 emission filter respectively, within holders 260 a,b and PMTadaptors 262 a,b. Main block 255 is closed by sealing plates 264 a,b andgaskets 266 a,b with fasteners 268

An electrical circuitry contains a microcontroller to control both thepulse rate to the LEDs 206 a,b and synchronize the readings from thePMTs 208 a,b. The LEDs 206 a,b are energized for a short time at afrequency of 100 Hz. At no time are both LEDs 206 a,b illuminated,eliminating some of the bleed through of the two fluorescent markers. Ahigh speed 16 bit analogue/digital converter is used to read the PMTs208 a,b and average the data. A laptop computer may be used for thesoftware component of this system, or the electrical circuitry,microcontroller, and software may be housed within the fluorescentdetector 200.

Mathematical Model

A two compartment mathematical model may be used to calculate GFR fromthe intensity ratio of the two tagged dextran molecules. This model maybe included in software which may be stored on an external computer orwithin the fluorescent detector 200. Alternatively, the mathematicalmodel may be hard wired circuitry either internal or external to theapparatus.

GFR and apparent volume of distribution can be measured by monitoringthe plasma disappearance of the fluorescently labeled dextran moleculeintravenously administered by a single dose bolus injection. FIG. 15illustrates a widely used two-compartment model, also known asthree-component model. The two compartments in question are vascularspace and interstitial space. The basic assumption for this model isthat the infused reporter molecule will distribute from the vascularspace to interstitial space after the bolus injection, but the markermolecule will be retained in the vascular space. The plasma removal ofthe reporter molecule only occurs from the vascular space.

The plasma clearance rate and the inter-compartment clearance rate aredenoted as G and k, respectively. The virtual volume for the vascularspace and interstitial space are V₁ and V₂, respectively. Asdemonstrated by Sapirstein et al. (Sapirstein, L, A, D, G, Vidt, et al.(1955). “Volumes of distribution and clearances of intravenouslyinjected creatinine in the dog.” American Journal of Physiology 181(2):330-6.) the amount change per unit time in V₁ is given by the followingequation:

$\begin{matrix}{{V_{1}\frac{C_{1}}{t}} = {{- {GC}_{1}} - {k( {C_{1} - C_{2}} )}}} & (2)\end{matrix}$

Total injected amount D can be expressed as the following:

D=C ₁ V ₁ +C ₂ V ₂ +G∫C ₁ dt  (3)

where C₁ and C₂ denote the concentrations of the reporter molecule inthe vascular and interstitial space, respectively.

Combining the two equations above yields the following second orderlinear differential equation (Sapirstein, Vidt et al. 1955):

$\begin{matrix}{{{V_{1}\frac{^{2}C_{1}}{t^{2}}} + {( {\frac{G + k}{V_{1}} + \frac{k}{V_{2}}} )\frac{C_{1}}{t}} + \frac{{kGC}_{1}}{V_{1}V_{2}}} = 0} & (4)\end{matrix}$

The general solution to equation (4) is a bi-exponential functionexpressed in equation (16) below:

C ₁(t)=Ae ^(−αt) +Be ^(−βt)  (5)

where the decay constants α and β can be expressed in k, G, V₁ and V₂(Sapirstein, L. A., D. G. Vidt, et al. (1955). “Volumes of distributionand clearances of intravenously injected creatinine in the dog.”American Journal of Physiology 181(2): 330-6.).

Assuming the inter-compartment movement is negligible before theintra-compartment mixing in V₁ is completed, then the following twoboundary conditions at t=0 become valid: C₀=DN/V₁ and C₂=0.

From equations (2), (3), (5), and the two boundary conditions we canderive the following (Sapirstein, L. A., D. G. Vidt, et al. (1955).“Volumes of distribution and clearances of intravenously injectedcreatinine in the dog.” American Journal of Physiology 181(2): 330-6.):

$\begin{matrix}{{GFR} = \frac{D}{{A/\alpha} + {B/\beta}}} & (6) \\{V_{1} = \frac{D}{A + B}} & (7) \\{V_{d} = \frac{D( {\frac{A}{\alpha^{2}} + \frac{B}{\beta^{2}}} )}{( {\frac{A}{\alpha} + \frac{B}{\beta}} )^{2}}} & (8)\end{matrix}$

where the total extracellular volume of distribution V_(d), is the sumof V₁ and V₂.

Parameters A, B, α, and β can be obtained by fitting the experimentaldata to equation (5).

In practice we may obtain V₁ using the marker molecule. If the linearrelationship between the concentration and fluorescence intensity holdsfor the reporter molecule, equation (5) can then be rewritten as:

F ₁(t)=A ₁ e ^(−αt) +B ₁ e ^(−βt)  (9)

where F₁ is the fluorescence intensity of the reporter molecule as afunction of time. A₁ and B₁ are constants.

Thus, equations (6) and (8) can be rewritten as follows:

$\begin{matrix}{{GFR} = \frac{V_{1}( {A_{1} + B_{1}} )}{{A_{1}/\alpha} + {B_{1}/\beta}}} & (10) \\{V_{d} = \frac{{V_{1}( {A_{1} + B_{1}} )}( {\frac{A_{1}}{\alpha^{2}} + \frac{B_{1}}{\beta^{2}}} )}{( {\frac{A_{1}}{\alpha} + \frac{B_{1}}{\beta}} )^{2}}} & (11)\end{matrix}$

where equation (10) represents GFR from intensity of a single, freelyfilterable reporter molecule type, and equation (11) represents thevolume distribution associated with a single, freely filterable reportermolecule type.

In addition, since the fluorescence of the marker is a constant overtime, equation (9) can be also expressed in terms of fluorescence ratioof the reporter molecule over the marker molecule. Thus, thebi-exponential equation becomes:

R(t)=A ₂ e ^(−αt) +B ₂ e ^(−βt)  (12)

where R(t) is the fluorescence ratio of the reporter molecule over themarker molecule.

Constants A₂, B₂, α, and β can be obtained by fitting the experimentdata to the above equation. Thus, the clearance GFR and the total volumeof distribution can be expressed as:

$\begin{matrix}{{GFR} = \frac{V_{1}( {A_{2} + B_{2}} )}{{A_{2}/\alpha} + {B_{2}/\beta}}} & (13) \\{V_{d} = \frac{{V_{1}( {A_{2} + B_{2}} )}( {\frac{A_{2}}{\alpha^{2}} + \frac{B_{2}}{\beta^{2}}} )}{( {\frac{A_{2}}{\alpha} + \frac{B_{2}}{\beta}} )^{2}}} & (14)\end{matrix}$

where equation (13) represents GFR from the intensity ratio between afreely filterable reporter molecule type and a larger marker moleculetype, and equation (11) represents the volume distribution associatedwith from a freely filterable reporter molecule type and a larger markermolecule type.

Evidently, when the inter-compartment volume exchange rate approacheszero, this model collapses to a single compartment model. However, ithas been shown that as the plasma clearance level increases thismono-exponential approximation will lead to an overestimation of the GFR(Schwartz, G. J., S. Furth, et al. (2006). “Glomerular filtration ratevia plasma iohexol disappearance: pilot study for chronic kidney diseasein children.” Kidney International 69(11): 2070-7; Yu, W., R. M.Sandoval, et al. (2007). “Rapid determination of renal filtrationfunction using an optical ratiometric imaging approach.” AmericanJournal of Physiology—Renal Physiology 292(6): F1873-80.)

Optical Catheter

Referring to FIGS. 16 and 17, the catheter 300 for use with the presentinvention is illustrated. The catheter 300 includes a fiber opticinsertion tool 304, a dual port luman 308, and a French 5 to 8 sizeintroducer 312. A standard 1 mm plastic fiber optical cable 316 may beinserted through a passageway 320 defined by a combination of theinsertion tool 304 joined with the luman 308 joined with the introducer312. Accordingly, each of these components is of a tubularconfiguration. Preferably, a 0.75 mm fiber optic cable is insertedthrough the passageway 320. The 0.75 mm diameter was only chosen toallow use of a standard 18 gauge introducer.

The insertion tool 304 includes a first tubular member 324 slidablewithin a second tubular member 328. Fluid-tight seals are provided onopposing ends of the second tubular member 328 by o-rings 332 about thefirst tubular member 324 and the fiber optic cable 316, respectively.The fiber optic cable 316 is securely held or fixed within the insertiontool 304 by a seal 336 at an opposite end of the insertion tool 304.

The insertion tool 304 is joined to one of the ports 340 a on the luman308. Homostatic seals 344 a,b are located on the ports 340 a,b. Theother port 340 b is to provide for bolus injection or a continuousinfusion of the fluorescent molecule. A luer connector 348 at anopposite end of the luman 308 joins the subject with the introducer 312.

The fiber optic cable 316 may comprise either single or multiple singlefibers for light delivery and collection of the emission and excitation.The fiber optic cable 316 is inserted within a subject's vein 352 bypressing the first tubular member 324 and the captive optical cable 316through the second tubular member 328 wherein the fiber optic cable 316is extensible from the catheter 300. The optical cable 316 traversesthrough the subject by the luer connector 348 through the introducer 312and into the subject's vein 352. The fiber optic cable 316 may have asmall permanent bend on an end inserted into the subject's vein 352.This bend helps penetrate the tissue and minimizes interference of thefiber optic cable 316 within the vein.

In use, the fiber optic cable 316 is an extension of, or placed incommunication with, the fiber optic cable 210 of the fluorescentdetector 200 to transmit a signal or signals generated at the subject'svein to the fluorescent detector 200 for evaluation.

The present invention discloses a unique and novel method and device forquantifying kidney function, but it also presents a unique method ofquantitatively determining liver function. For example, a dyecomposition of a larger molecular weight marker and smaller molecularweight reporter molecules is injected into a subject, and the ratio ofthe decrease of the reporter molecule to the marker molecule is used todetect kidney function. The smaller reporter molecules are filtered bythe kidney while the marker molecules are remained in the vascularsystem. For a reasonable ratio of marker molecules to signal moleculesto be detected, the marker molecule must remain in the blood atrelatively consistent levels during the diagnostic test. Eventually on amuch longer time scale (typically 12 to 24 hours) the marker moleculewill typically be absorbed and processed from the vascular system by theliver instead of the kidney. Here a novel method and device aredescribed, where relative liver function and health may bequantitatively determined by measuring the absolute decrease of themarker molecule in the blood over time. This method will have advantagesover other methods by providing a quantitative value on an arbitraryscale that correlates to liver health. As a result, medical careprofessionals will be provided with a new tool allowing them to bettertreat their patients and predict proper dosing of certain drugs. Thismethod would use the same device as described previously, and would alsoutilize the same dye composition as described previously; however, itwould provide a method of analyzing the results to provide additionalfunction and utility using the following equation:

$\begin{matrix}{{LiverFunction} = \frac{{Emission}\mspace{14mu} {from}\mspace{14mu} {Marker}\mspace{14mu} {Molecule}}{time}} & (15)\end{matrix}$

While the specific embodiments have been illustrated and described,numerous modifications come to mind without significantly departing fromthe spirit of the invention, and the scope of protection is only limitedby the scope of the accompanying Claims.

1.-28. (canceled)
 29. A method of determining glomerular filtration rateof a kidney of a mammalian subject, the method comprising: providing asource of fluorescent reporter molecules and fluorescent markermolecules; introducing said reporter molecules and said marker moleculesinto a vascular system of said mammalian subject; measuring fluorescenceintensity of said reporter molecules and said marker molecules withinsaid vascular system; determining a ratio of intensity of saidfluorescence intensity of said measured reporter molecules and saidmeasured marker molecules; and calculating glomerular filtration rate ofsaid kidney.
 30. The method of claim 29, wherein said glomerularfiltration rate of said kidney is calculated according to equation:${GFR} = \frac{V_{1}( {A_{2} + B_{2}} )}{{A_{2}/\alpha} + {B_{2}/\beta}}$wherein V₁ is plasma volume, A₂ and B₂ are constants; α is a fast phasedecay constant, and β is a slow phase decay constant; wherein said ratioof intensity of said reporter molecules and said marker molecules iscalculated at predetermined intervals; and wherein said constants A₂,B₂, α, and β are calculated by fitting the ratio intensity data toequation:R(t)=A ₂ e ^(−αt) +B ₂ e ^(−βt) where R(t) is the ratio of intensity asa function of time.
 31. The method of claim 29, wherein said glomerularfiltration rate of said kidney is calculated according to equation:${GFR} = \frac{D}{C_{2}( {{A/\alpha} + {B/\beta}} )}$wherein D is a volume of reporter molecules, C₂ is a plasmaconcentration of a marker molecules, A and B are constants, α is a fastphase decay constant, and β is a slow phase decay constant.
 32. Themethod of claim 29, wherein said glomerular filtration rate of saidkidney is calculated according to equation:$V_{d} = \frac{{V_{1}( {A_{2} + B_{2}} )}( {\frac{A_{2}}{\alpha^{2}} + \frac{B_{2}}{\beta^{2}}} )}{( {\frac{A_{2}}{\alpha} + \frac{B_{2}}{\beta}} )^{2}}$wherein V_(d) is total extracellular volume, V₁ is a virtual volume of avascular space, A₂ and B₂ are constants, α is a fast phase decayconstant, and β is a slow phase decay constant.
 33. The method of claim29, wherein said reporter molecules are filtered by the kidney, and saidmarker molecules are retained in the vascular system.
 34. The method ofclaim 29, wherein said reporter molecules and said marker molecules arechemically stable in the vascular system during measurement of kidneyfunction.
 35. The method of claim 29, wherein said reporter moleculesand said marker molecules are fluoresceins.
 36. The method of claim 29,wherein said reporter molecules and said marker molecules comprisedextrans.
 37. The method of claim 29, wherein said marker molecules havea greater molecular weight than said reporter molecules.
 38. The methodof claim 29, wherein said reporter molecules have a molecular weightbetween 1 kDa and 70 kDa.
 39. The method of claim 29, wherein saidmarker molecules have a molecular weight greater than 100 kDa.
 40. Themethod of claim 29, wherein said reporter molecules have a firstfluorescent characteristic and said marker molecules have a secondfluorescent characteristic, and wherein said first fluorescentcharacteristic and said second fluorescent characteristic aredistinguishable.
 41. The method of claim 42, wherein said firstfluorescent characteristic is a first excitation wavelength and a firstemission wavelength and said second fluorescent characteristic is asecond excitation wavelength and a second emission wavelength, saidfirst and second fluorescence excitation wavelengths and said first andsecond fluorescence emission wavelengths being unequal.
 42. The methodof claim 29, wherein the step of introducing is performed using acatheter.
 43. The method of claim 29, wherein said step of measuring isperformed using an optic fiber in communication with a detector.